1. Field of the Invention
The DegP (HtrA) protease is a multifunctional protein essential for the removal of misfolded and aggregated proteins in the periplasm. DegP has been shown to be essential for virulence in several Gram negative pathogens. Only three natural targets for DegP have been described: colicin A lysis protein (Cal), pilin subunits (K88, K99, Pap) and recently HMW1 and HMW2 from Hemophilus influenzae. In vitro, DegP has shown weak protease activity on casein and several other non-native substrates. The present inventors have identified the major pilin subunit of the Pap pilus, PapA, as a native DegP substrate and demonstrated binding and proteolysis of this substrate in vitro. Using an NH.sub.2 -terminal affinity tag the present inventors have purified PapA away from the PapD chaperone, in the presence of denaturant, to use as a proteolysis substrate. This finding will allow the identification of the DegP recognition and cleavage sites in substrate proteins, and further, allow the design of small molecule inhibitors of protease function.
2. Description of the Related Art
Proteolysis of misfolded and denatured proteins in the bacterial cytoplasm and periplasm is a crucial housekeeping function and critical for cell viability (Pallen, M. J. and Wren, B. W. (1997) Mol. Microbiol. 26,209-221; Miller, C. G. (1996) in Escherichia coli and Salmonella Cellular and Molecular Biology (Neidhardt, F. C., eds) pp. 938-954, ASM Press Washington D.C.). It is becoming increasingly clear that the proteolytic machinery is also an essential component for bacterial pathogenesis (Pallen & Wren, supra). Recently, scientists have uncovered a regulatory system, CpxA/CpxR, that responds to the changing environment of the periplasm; recruiting proteases, chaperones and "foldases" to assist in managing the state of affairs in this bacterial compartment (see, e.g., Danese, P. N., et al. (1995) Genes and Development 9,387-398; Danese, P. N. and Silhavy, T. J. (1997) Genes Dev. 11, 1183-1193). As the host often presents a hostile environment to the invading organism it is suggested that the CpxA/CpxR regulatory circuit is "tripped" upon engaging the host defenses. One of the most important proteases in the periplasm, the DegP/HtrA serine protease (Pallen & Wren, supra) is a member of the CpxA/CpxR regulon (Danese, P. N., et aL (1995), supra; Danese and Silhavy (1997) supra). This protein is also a key player in pathogenesis in Salmonella, Brucella, and Yersinia (Pallen & Wren, supra). Specifically, DegP has been shown to be a virulence determinant in Salmonella typhimurium, Brucella abortus and Yersinia enterocolitica. According to the current model of DegP function in pathogenesis, DegP acts to remove misfolded proteins and protein aggregates that result from exposure to reactive oxygen intermediates in the host. In the absence of functional DegP, these protein aggregates compromise the pathogenic process (Pallen & Wren, supra).
The DegP (degradation) nomenclature refers to the initial mapping of a mutation in E. coli that allowed the accumulation of unstable fusion proteins in the periplasm (Strauch, K. L., Johnson, K. and Beckwith, J. (1989) J. Bacteriol. 171,2689-2696; Strauch, K. L. and Beckwith, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,1576-1580). The HtrA (heat shock regulated) designation indicates that a transposon insertion in the same gene resulted in a heat sensitive growth phenotype (Lipinska, B., Sharma, S. and Georgopoulos (1988) Nucleic Acids Research 16,10053-10066). Lastly, DegP was also designated protease Do, again as a mutation that conferred a heat sensitive phenotype in E. coli (Seol, J. H., et al. (1991) Biochemical and Biophysical Research Communications 176,730-736). DegP exhibited functional protease activity in in vitro assays using casein as a substrate, although its activity on this substrate was weak (Lipinska, B., Zylicz, M. and Georgopoulos, C. (1990) J. Bacteriol 172,1791-1797). Lipinska et al. demonstrated that the activity on casein was inhibitable by DFP and not by any other known protease inhibitors, suggesting that DegP is a serine protease. Site directed mutagenesis at serine 210 and histidine 105, two components of the serine protease catalytic triad, compromised DegP function in vitro and in vivo; i.e. strains carrying serine 210 or histidine 105 mutant derivatives were sensitive for growth at elevated temperatures (Skorko-Glonek, J., et al. (1995) Gene 163,47-52). The preferred substrate for DegP appears to be proteins that are globally or transiently denatured; suggesting that the role in vivo is to clear misfolded or denatured proteins from the periplasm (Kolmar, H., Waller, P. R. H. and Sauer, R. T. (1996) J. Bacteriol 178,5925-5929). In support of this finding, Laskowa et al. (Laskowska, E., et al. (1996) Mol Microbiol. 22,555-571) demonstrated in vitro that purified DegP protein would degrade thermally aggregated proteins fractionated from E. coli extracts and that the DnaJ chaperone would antagonize DegP degradation; i.e. the chaperone would aid in refolding the proteins such that they were no longer targets for degradation by DegP.
In addition to its weak protease activity, DegP/HtrA has been shown to be a virulence factor for several pathogenic organisms. In Salmonella typhimurium, htrA nulls were found to be avirulent and more susceptible to oxidative stress (Johnson, K., et al. (1991) Mol. Microbiol. 5,401-407). The authors of this study suggest that the htrA mutants are less able to withstand oxidative killing within the macrophage. An htrA lesion was found to be useful in attenuating Salmonella typhi for implementation as a vaccine strain. Similarly, Brucella abortus and Brucella melentensis htrA null mutants were attenuated for virulence in goats and found to be significantly more sensitive to oxidative killing by cultured neutrophils in vitro (Elzer, P. H., et al. (1996) Research in Veterinary Science 60,48-50; Elzer, P. H., et al. (1 996) Infection and Immunity 64,4838-4841; Phillips, R. W., et al. (1997) Research in Veterinary Science 63,165-167). An isogenic pair, wild-type and htrA null mutant, in Yersinia enterocolitica were created and tested in a mouse yersiniosis model. HtrA was found to be essential for virulence and the mutant strain was more sensitive to oxidative stress (Li, S.-R., et al. (1996) Infection and Immunity 64,2088-2094). Finally, Boucher et al. ((1996) J. Bacteriol. 178,511-523) recently demonstrated that Pseudomonas aeruginosa conversion to mucoidy, the so-called CF phenotype involves two HtrA homologs. DegP homologs have been found in Streptococcus pneumoniae (Gasc, A-M et al. (1998) Microbiology 144:433-439), Streptococcus pyogenes, and Staphylococcus aureus. All three homologs share the catalytic triad of the E. coli DegP protein.
The first identified in vivo target for DegP was colicin A lysis protein (Cal) (Cavard, D., Lazdunski, C. and Howard, S. P. (1989) J. Bacteriol. 171,6316-6322). DegP was found to degrade the acylated precusor form of Cal into two fragments. Mature Cal also accumulated to higher levels in degP mutant strains (Cavard et al.(1989), supra). A second family of DegP targets was identified as bacterial pilins. The K88 and K99 pilin subunits were found to accumulate to higher levels in degP mutant strains (Bakker, D., et a. (1991) Mol. Microbiol. 5,875-886). A more detailed study of this phenomenon demonstrated that P pilins, specifically PapA, are substrates for the DegP protease (Jones, C. H., et aL (1997) EMBO J. 16,6394-6406). More recently the H. influenzae non-pilus adhesin proteins HMW1 and HMW2 were found to be in vivo substrates for DegP (St. Geme III, J. W. and Grass, S. (1998) Mol. Microbiol 27,617-630).
The DegP/HtrA sequence was published in 1988 (Lipinska, Sharma, & Georgopoulos, supra). HtrA is one of several dozen proteases in E coli and is known to have homologs in cyanobacteria, mycobacteria, yeast and man (Pallen & Wren, supra). There are also two homologs of DegP: DegQ and DegS in E coli (Kolmar et al. (1996), supra; Waller, P. R. and Sauer, R. T. (1996) J. Bacteriol 178,1146-1153). A new homology region has recently been identified in DegP that is conserved in many eukaryotic proteins (Pallen & Wren, supra). Downstream from the catalytic sequence-208GNSGGAL214 are two PDZ domains (Levchenko, I., et al. (1997) Cell 91,939-947). These 80-100 amino acid domains are found in nearly 100 proteins, mostly eukaryotic, and probably play roles in protein-protein interactions, either facilitating multimer formation or substrate binding (Levchenko, I., et al. (1997), supra). The PDZ domain homology is maintained in the recently identified Gram-positive DegP homologs. Interestingly, Kolmar et al. (1996, supra) recently demonstrated that DegP forms dodecamers in vitro, although it remains to be seen if the PDZ domains contribute to DegP multimerization. If DegP does function as a multimer in vivo it would be reminiscent of the proteosome machines described in eukaryotic cytosol and ER (Pallen & Wren, supra).
Early in vivo data suggested that pilins were DegP substrates (Bakker, D., et al. (1991) supra). Expression of pilin subunit proteins in the absence of the chaperone resulted in failure to accumulate subunit in the periplasm and degP mutant strains accumulated more subunit in the periplasm (Bakker, D., et al. (1991), supra; Jones, et al. (1997) supra; Hultgren, S. J., Normark, S. and Abraham, S. N. (1991) Annu. Rev. Microbiol 45,383-415; Hultgren, S. J., Jones, C. H. and Normark, S. N. (1996) in Escherichia coli and Salmonella; Cellular and Molecular Biology (Neidhardt, F. C., eds) pp. 2730-2756, ASM Press Washington D.C.). Moreover, subunit expression in the degP mutant was highly toxic (Jones et al. (1997), supra). Both the toxicity and accumulation was suppressed by complementation with degP (Jones et al. (1997), supra. A significant obstacle to the study of pilus biogenesis is the inability to purify subunits in the absence of the PapD chaperone. This was overcome by the addition of an affinity tag to the amino-terminus of PapA. This provided for the purification of large quantities of PapA under denaturing conditions. Renaturation of PapA in the presence of the PapD chaperone allowed the formation of the PapD-PapA complex. Moreover, mixing DegP with denatured PapA resulted in affinity purification of a PapA-DegP complex and proteolysis with release of an amino-terminal PapA fragment.